1. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Micro 20 Lab 2- Microscopy

2. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Microscopy: The Instruments  A simple microscope has only one lens van Leeuwenhoek. Figure 1.2b

3. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings The compound light microscope 1 of 66 : 03- 01_Microscope_L.j pg

4. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings The path of light  In a compound microscope, the image from the objective lens is magnified again by the ocular lens.  Total magnification = objective lens  ocular lens Figure 3.1b

5. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Magnification  Objective lens X Ocular Lens= magnification 4X 10X = 40X 20X 10X = 200X 40X 10X = 400X 100X* 10X = 1000X 100X lens requires oil immersion.

6. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Microscopy: The Instruments  Resolution: The ability of the lenses to distinguish two points.  A microscope with a resolving power of 0.4 nm can distinguish between two points ≥ 0.4 nm.  Shorter wavelengths of light provide greater resolution.  The resolving power of light microscope is 0.2  m.

7. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Refraction using an oil objective lens  Refractive index: The light-bending ability of a medium.  The light may bend in air so much that it misses the small high- magnification lens.  Immersion oil is used to keep light from bending. Figure 3.3

8. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Brightfield Illumination  Dark / stained objects are visible against a bright background.  Light reflected off the specimen does not enter the objective lens.  Shows internal structures Figure 3.4a–b

9. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Darkfield Illumination  Light objects are visible against a dark background.  Live unstained specimen  Light reflected off the specimen enters the objective lens. Figure 3.4a–b

10. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Phase-Contrast Microscopy  Accentuates diffraction of the light that passes through a specimen.  Used for colorless or transparent specimen Figure 3.4c

11. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Use of Microscope:  Preparation of specimen:  Wet mount - specimen in liquid environment under a glass cover.  Stained- specimen colored with chemicals to be easily visible

12. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Use of Microscope:  Observation of specimen:  Place specimen on clean glass slide  Place slide on stage and rotate the 10x objective into place  Focus on image of object for maximum clarity  Rotate the 40x objective lens into place and view again for higher magnification of image.  Record observations and remove slide from stage  Discard appropriately

13. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Care of Microscope:  Always carry microscope carefully (with 2 hands – one under the base and one on the arm) and place gently.  Clean the lens with lens paper only – start from lower power to higher power objectives  Lock the microscope lockers after placing microscopes inside when finished

14. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Important terms  Magnification: The number of times the image of the object is enlarged  Resolution: The ability of the microscope to provide detail and clarity  Resolving Power (Rp): The smallest distance two closely adjacent points have to be separated in order to be distinguished as separate entities  Lambda: The speed at which light rays travel (measured in nm)

15. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Important terms  Numerical Aperture (NA): property of the objective lens to gather light.  Refractive index (n): The amount of refraction (bending of light rays) when it passes through a medium.  Inter-pupillary distance: The distance between the 2 oculars.  Working distance: The distance between objective and specimen  Field: The area seen when looking through the oculars.  Parfocal: The phenomenon of partial focus when switching objectives.